Parvoviruses are icosahedral, single stranded, DNA viruses. Their genome is about 5.1 kilobases and includes the P4 and P38 promoters, which drive the expression of non structural (NS1 and NS2) and structural (VP1 and VP2)) proteins, respectively. Parvoviruses belong to the Parvovirinae family, together with Dependo- and Erythroviruses. The Dependoviruses include the adeno-associated viruses (AAV). These viruses, as the name suggests, are dependant on a helper virus, like adenovirus, for a productive infection cycle [1]. On the other hand, Erythroviruses and Parvoviruses are autonomous; they are able to complete their productive cycle without a helper virus. Erythroviruses include B19, known to be pathogenic to humans.
Rodent parvoviruses are non-pathogenic for humans and show oncotropic and oncolytic properties that make them very attractive agents for cancer therapy. To further enhance their intrinsic anti-neoplastic ability, recombinant viruses have been generated to deliver anti-tumour transgenes to cancer cells. The strategy consists of replacing the VP capsid genes with a particular transgene [2]. The vectors retain the NS1/2 coding sequence (under the control of the parvoviral P4 promoter) and the parvoviral genome telomeres which are necessary for viral DNA amplification and packaging. The transgene of interest is generally expressed under the control of the P38 promoter, which is strongly trans-activated by the NS1 protein. Production of the recombinant viruses can only occur in producer cells, providing the VP proteins in trans. The deletion of the capsid coding region in the viral genome gives the opportunity to insert transgenes up to approximately 2500 nt, although large VP deletions reduce production titers significantly, probably due to the suppression of important virus packaging cis-acting elements [2]. The recombinant parvoviral vectors generated in this way are replication-defective, providing an additional therapeutic safety advantage in comparison to their replicative counterparts. Examples of transgenes successfully transduced by recombinant parvoviruses include toxic genes, such as herpes simplex virus thymidine kinase (e.g. used in combination with the prodrug ganciclovir) [3] or Apoptin [4]. Virions expressing these transgenes have improved anti-neoplastic activities in comparison to wild type viruses. Recombinant parvoviruses encoding immunomodulatory genes such as cytokines or chemokines, like Interleukin-2 (IL-2) or Monocyte Chemotactic Protein 1 or 3 (MCP-1) [5-8], Interferon-γ-inducible protein (IP-10) [9] have been also generated. These virions induce potent antitumoral bystander effects in various in vitro and in vivo tumor models. Recently, antitumoral synergistic effects were observed using a combination of recombinant viruses transducing Tumor Necrosis Factor-α (TNF-α) and IP-10 [10].
The helper activities of adenovirus towards AAV replication have been studied extensively. The adenoviral E1A protein is essential for the activation of AAV gene expression by binding and activating the AAV-2 P5 rep promoter [12]. Similarly, E2A, another adenoviral protein, activates the AAV-2 PS promoter transcription [13]. E2A also appears to cooperate with virus associated RNA I (VAI RNA) to enhance the translation of AAV-2 RNAs [14]. The adenoviral E4 (orf6) enhances the conversion of single-stranded recombinant AAV-2 genomes into double-stranded genomes, a rate-limiting step of viral DNA-replication both in vitro and in vivo [15]. VAI RNA can also support AAV-5 replication. It has been described that VAI RNA physically interacts with the double-stranded RNA-activated protein kinase (PKR), which would otherwise elicit an antiviral immune response blocking viral protein production [16].
Studies with recombinant AAV-2 have confirmed that a small subset of adenoviral genes is sufficient for the helper effect. In HEK 293 cells, which provide the E1 gene in trans, the minimal set of genes for efficient recombinant AAV-2 production is E2a, E4 (orf6) and the VAI RNA gene [17, 18]. A helper plasmid named pXX6, containing this set of genes, is currently used for the production of adenovirus-free recombinant AAV-2 [18]. This plasmid sustains AAV-2 production in a similar way as infectious adenovirus [18, 19]. Adenovirus genes (E2a, E4orf6, and VAI RNA genes) also allow human erythrovirus B19 DNA replication and viral production in HEK 293 cells, that would otherwise be non-permissive for B19 [20].
The interaction between adenovirus and autonomous parvoviruses has however remained less explored. H-1 parvovirus replication in secondary cultures of normal human embryonic lung cells is not initiated, unless they are co-infected with adenovirus serotype 12 as a helper virus [21]. In addition, adenovirus serotype 2 induces an increase of MVMp parvovirus DNA replication and relocalizes MVMp DNA from host nucleoli to adenovirus replication factories in HeLa cells (22].
One major challenge in the development and optimization of recombinant parvoviruses (rec.PVs) for clinical applications is to increase the amount of viruses being produced. Due to their non-proliferative nature, their production depends solely on the transfection efficiency of the parvoviral genomic components into the packaging cell lines (normally human embryonic kidney cells, HEK 293 or 293T). Also the use of packaging cell lines constitutively expressing the VP proteins did not significantly improve the production of recombinant parvoviruses [11]. It remains of high importance to develop means to increase recombinant parvovirus production.
Thus, the problem on which the present invention is based was to provide a means for increasing the efficiency of recombinant parvovirus production.
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.